Keratinocytes are the major cell type in the epidermis, a multilayered group of cells without blood vessels on the surface of the skin. Presumably, proliferation and growth of epidermal cells in vivo is nourished by nutrients from the dermal layer, including growth factors. Keratinocytes have been propaged in vitro in continuous culture using defined medium for example as described in Shipley, G.D., et al., Arch Dermatol (1987) 123:1541A-1544A. The medium contains various inorganic ions, trace elements, amino acids, vitamins and other organic compounds and is not qualitatively different from defined media used to culture other mammalian cell lines. In this publication it was noted that insulin and epidermal growth factor (EGF) were required for clonal growth in vitro and that hydrocortisone was beneficial.
Basic fibroblast growth factor (bFGF) is able to replace EGF in supporting the clonal growth of keratinocytes in a dose-dependent manner; acidic fibroblast growth factor (aFGF) is also a potent mitogen (Shipley, G.D., et al., J Cell Physiol (1989) 138:511-518, incorporated herein by reference). These FGFs are members of a class of heparin-binding growth factors (HBGF) which includes the products of several potential oncogenes. The ability of bFGF to stimulate proliferation of keratinocytes is inhibited by heparin; heparin has no effect on stimulation by EGF or TGFa and only minimal effect on stimulation by aFGF.
It is also known that transforming growth factors (TGF) are produced by virally transformed cells and tumor cell lines. These factors stimulate soft agar colony formation of cells that are normally anchoragedependent and cause the overgrowth of these cells in a monolayer culture. Two major classes of transforming growth factors have been identified--TGF--alpha and TGF--beta. Other growth factors including platelet-derived growth factor (PDGF) and HBGFs also can contribute to anchorage-independent growth of some target cells. Shipley, G.D., J Tissue Culture Methods (1986) 10:117-123, describes a simple in vitro serum-free thymidine-incorporation assay for detection of transforming growth factors.
A purified factor from fibroblasts designated keratinocyte growth factor (KGF) was described by Rubin, J.S., et al., Proc Natl Acad Sci USA (1989) 86:802-806. This factor is labile to acid treatment and stable to reduction. Other growth factors which have been described are transforming growth factor type-e (Halper et al., Cancer Res (1987) 47:4552-4559), and DNA synthesis factor (Masuda et al., In Vitro Cell and Devel Biol (1988) 24:893-899; Masuda et al., Cell Biol Int Rep (1987) 1:359-365).
Shoyab, M., et al., Proc Natl Acad Sci USA (1988) 85:2528-2532, described a glycoprotein which they termed amphiregulin (AR) which inhibits the growth of human carcinoma cells in culture but stimulates proliferation of human fibroblasts and other tumor cells. Amphiregulin was obtained from serum-free conditioned medium of the MCF-7 cell line derived from a human breast carcinoma which was stimulated with phorbol 12-myristate 13-acetate. Unlike keratinocyte growth factor (KGF), AR was not acid labile and the foregoing report indicated that the glycosylation could be removed but activity retained, and the N-terminal sequence was disclosed. In a subsequent paper, Shoyab, M., et al., Science (1989) 243:1074-1076, the complete amino acid sequence of a "truncated" form containing 78 amino acids as determined using protein-sequencing techniques as well as a larger form containing 6 additional amino acid sequences at the N-terminal end was reported. It was further disclosed that amphiregulin binds to the EGF receptor but less strongly than does EGF itself. Amphiregulin was shown to stimulate the growth of the murine keratinocyte cell line Balb/MK and could supplant the EGF requirement in these cells. In these assays, the keratinocytes were plated at 10.sup.4 cells per well and cultured overnight to a starting cell density of approximately 10.sup.4 cells/cm.sup.2.
In a more recent publication, Plowman, G.D., et al., Mol Cell Biol (1990) 10:1969-1981, this same group reported the cloning of the gene encoding the 252 amino acid precursor of the 84 amino acid amphiregulin "mature" peptide secreted by MCF-7 cells, which precursor had an organization similar to the transmembrane precursor corresponding to TGF-alpha. Human placenta and ovaries also were reported to express significant amounts of the AR transcript. Assessment of the biological activity of the amphiregulin was not elaborated over that of the Science paper which had shown only data that AR had stimulatory activity with respect to murine keratinocytes under the high cell density conditions of the reported assay. These cells do not make KAF.
The reported amino acid sequence of amphiregulin matches that of the keratinocyte autocrine factor of the present invention. These data are also the subject of published British application GB2214185A, published 31 August 1989.
Two types of models have been described to account for the loss of growth control in tumor cells. In one, the "autocrine hypothesis," the production of and response to a growth factor by the same cells, leading to an increase in proliferation rate of these cells, is postulated. In an alternative "paracrine hypothesis," secretion of factors by the tumor cells are thought to stimulate the production of growth factors by surrounding tissues, which, in turn, stimulate the growth of the tumor cells.
In short, while the process whereby cells are permitted to proliferate in vivo and in vitro is far from understood, it is known that there is a complex interrelationship of growth factors, which in some way regulates this proliferation. The present invention relates to an autocrine factor selective for the proliferation of keratinocytes.